The aged nursing home patient is frequently incontinent of urine. A common management technique for this patient is the indwelling urinary catheter which, when left in place for more than 30 days, in universally associated with polymicrobial bacteriuria. A bacterial library of 7639 retrievable isolates was established from collection of weekly urine specimens from 51 aged chronically catheterized nursing home patients. Bacteria were speciated, assigned a storage number which was entered into an IBM 4341 file, and stored at -70 degrees C. Nearly half (47%) of the gram-negative bacilli isolated produced the enzyme urease which has been implicated in pyelonephritis, hepatic coma, hyperammonemia, complement inactivation, and kidney and bladder stone formation. The genes encoding urease were cloned from a strain representing the most prevalent isolate, Providencia stuartii (1049 isolates) into a high copy number plasmid vector. Limited reports characterizing ureases from different bacterial species isolated from the urinary tract provide conflicting data and fail to provide a clear concept of the relatedness of heterogeneity of these enzymes. In addition, tests of urease as a virulence factor in animal models of urinary tract were performed in such a manner that urease may not have been the only factor varied. The specific aims of this project are to systematically classify the urease of bacterial species isolated from the catheterized urinary tract including Providencia stuartii Proteus mirabilis, Morganella morganii, Klebsiella pneumoniae, and Providencia rettgeri with respect to DNA homology, molecular weight, subunit composition, metal ion requirements, kinetics of urea hydrolysis, and inhibition by urease inhibitors; and to determine the contribution of urease as a virulence factor in a mouse model of urinary tract infection by using bacterial strains specifically deleted (or not deleted) for urease encoding sequences. Methodology will include standard recombinant techniques for cloning and deletion analysis; and Tn5 mutagenesis. Genetic probes will be prepared by nick translation and hybridized to colony, dot, and Southern blots. Urease will characterized on denaturing and nondenaturing polyacrylamide gels, by column chromatography, and isoelectric focusing. Enzyme activities will be determined by potentiometric and spectrophotometric assays. Urease-negative bacterial strains will be constructed by marker exchange and tested in a CBA mouse model of urinary tract infection.